Image Quantification

TIFF images of both channels (Cy3 and Cy5) from the microarray are imported into, and processed by, a piece of image analysis software such as Imagene (http://www.biodiscovery.com/imagene.asp) or BlueFuse (http://www.cambridge-bluegnome.com/products/bluefuseformicroarrays/index.htm). The image analysis software programs perform the laborious task of measuring the fluorescence intensities of many thousands of spots at once, which they do by superimposing a malleable grid over the array surface, from which it detects and quantifies the median intensity of the pixels within each spot as well as within a local background area. The median is used because it is not strongly influenced by outliers, and is therefore a robust estimate of the mean. The software links the spot coordinates to an internal database that contains details of all of the genes represented by the spots and produces a tab-delimited or XML results file. Included in this file are the spot coordinates, gene names and identifiers, and the mode and median of the fluorescence readings as well as information about the background reading, spot size, and many other pieces of supplementary information. Usually a separate file for each of the Cy dyes is produced, meaning that an array experiment will produce many large files that contain huge amounts of data. However, the format of this data file means it can easily be imported into any data analysis software package for processing.